1. Volume 27, Issue 6, Pages 871-884 (December 2007)
Regulation of T Cell Receptor β Gene Rearrangements and Allelic Exclusion by the Helix-Loop-Helix Protein, E47 
Yasutoshi Agata, Nobuyuki Tamaki, Shuji Sakamoto, Tomokatsu Ikawa, Kyoko Masuda, Hiroshi Kawamoto, Cornelis Murre 
Immunity 
Volume 27, Issue 6, Pages (December 2007)
DOI: /j.immuni
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 E47-Dosage-Dependent Regulation of TCRβ Gene Rearrangement
(A) Flow-cytometric analysis of E47-deficient thymocytes. Thymocytes prepared from E15 E47+/+, E47+/Δ, or E47Δ/Δ mice were analyzed for surface expression of CD44 and CD25 on lineage- (CD3, CD4, CD8, and DX5) negative cells.
(B) Schematic representation of the mouse TCRβ locus. Vβ, Dβ, Jβ gene segments, constant (Cβ) genes, and the enhancer (Eβ) are indicated. A diagram of the PCR analysis to detect Vβ-DJβ and Dβ-Jβ rearrangements is also shown. Arrows indicate PCR primers.
(C) PCR analysis of TCRβ rearrangement in E47-deficient thymocytes. Genomic DNAs prepared from E47+/+, E47+/Δ, or E47Δ/Δ E15 fetal thymocytes were analyzed for Vβ-DJβ1, Vβ-DJβ2, Dβ1-Jβ1, Dβ1-Jβ2, and Dβ2-Jβ2 rearrangements by PCR with Vβ or Dβ upstream primer (Vn, D1, or D2) in conjunction with Jβ1 or Jβ2 downstream primer (J1 or J2) and then Southern blotting. PCR products amplified from the Dβ1-Jβ1 and Dβ2-Jβ2 germline sequences are indicated by arrowheads (GL). Equal DNA quantities were verified by PCR of the Cd3e gene with 4-fold serially diluted DNA samples visualized by ethidium bromide gel staining. Data are representative of two independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Decreased E47 Protein Amounts and E Box Binding Activity by the E47 Heterozygosity
(A) Immunoblot analysis of E47-deficient thymocytes. Two-fold serial dilutions of total cell lysates prepared from E47+/+Rag2−/−, E47+/ΔRag2−/− and E47Δ/ΔRag2−/− adult thymocytes were analyzed for E47, HEB, and α-tubulin. Intensities of specific bands were measured with a densitometer, and relative intensities to E47+/+Rag2−/− thymocytes are indicated as numbers.
(B) EMSA analysis of E47-deficient thymocytes. Whole-cell extracts prepared from the thymocytes as in (A) were preincubated with control IgG, anti-E47, or anti-HEB and analyzed for binding activity to the μE5 probe. The same samples were tested for the Oct probe as a loading control. Arrowheads indicate specific complexes. Intensities of specific bands were measured with a densitometer, and relative intensities to E47+/+Rag2−/− thymocytes are indicated as numbers and graphs. Data are representative of two independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Decreased Vβ chromatin Accessibility in E47 Heterozygous Thymocytes
(A) Flow-cytometric analysis of E47-deficient thymocytes. E47+/+Rag2−/− and E47+/ΔRag2−/− adult thymocytes were analyzed for surface expression of CD4, CD8, CD44, and CD25 on live-gated cells (upper) and CD44 and CD25 on lineage-negative cells (Lin: CD3, CD4, CD8, and DX5) (lower).
(B) RT-PCR analysis of E47-deficient thymocytes. Vβ and Dβ germline transcript levels in E47+/+Rag2−/− (black bars) or E47+/ΔRag2−/− (gray bars) thymocytes were quantified by real-time PCR. Each transcript expression was normalized to β-actin mRNA, and relative transcript expression to that of E47+/+Rag2−/− thymocytes is shown as the mean of duplicate PCRs with range. Ptcra (pTα) and Rag1 (RAG1) transcript expression was also analyzed. Data are representative of two independent experiments.
(C) ChIP analysis of histone H3 acetylation in E47-deficient thymocytes. Chromatin prepared from E47+/+Rag2−/− (black bars) or E47+/ΔRag2−/− (gray bars) thymocytes was immunoprecipitated with anti-acetylated histone H3 (AcH3). The bound and input fractions were quantified by real-time PCR, and ratios of bound versus input are indicated as a percentage of input for each DNA segment. Data are the mean of duplicate PCRs with range and are representative of three independent experiments. Dβ1U, Dβ2U and Dβ1D, Dβ2D indicate upstream and downstream region of the Dβ1 or Dβ2 gene segment, respectively. Eβ indicates the TCRβ enhancer. Vβ14S and P indicate RSS and promoter region of the Vβ14 gene segment, respectively.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 ChIP-on-Chip Analysis of E2A Binding in the TCRβ Locus
Custom microarrays containing 50-mer probes that tiled through the entire mouse TCRβ locus were hybridized with amplicons prepared from E2A and control IgG ChIP DNA and an input DNA. The hybridization intensity signals were normalized across the entire array, by the taking of the ratio of the medians for all features and the normalization of them to unity. After normalization, the ratio was calculated by the division of the E2A or IgG hybridization intensity signal by the input control signal for each oligonucleotide probe and is shown as log2 ratio. Shown are the representative data in the forward strand among duplicates in each forward and reverse strand with similar results.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Modulation of E2A, CBP, and Histone H3 Acetylation in the TCRβ Locus by Pre-TCR Signaling
ChIP analysis of E2A, CBP, and histone H3 acetylation in total thymocytes from untreated adult Rag2−/− mice (black bars) and Rag2−/− mice injected with anti-CD3ɛ 5 days before preparation (white bars). Chromatin was immunoprecipitated with antibodies directed against either E2A or CBP or acetylated histone H3 (AcH3) or with control IgG. The bound and input fractions were quantified by real-time PCR, and ratios of bound versus input are indicated as a percentage of input for each DNA segment. Data are the mean of duplicate PCRs with range and are representative of three independent experiments. The abbreviations defined in Figure 3C are used. AcH3 in the Vβ portion are shown in a magnified scale for the clarification of the effects of anti-CD3 treatment.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
Enforced E47 Expression Overrides Allelic Exclusion in TCRαβ Transgenic Thymocytes
(A) PCR analysis of Vβ-DJβ and Dβ-Jβ coding joints in cultured thymocytes. Total thymocytes prepared from E15 wild-type (WT) and DO11.10 TCRαβ transgenic (Tg) mice were transduced with control virus (Mock) or E47-ER-expressing virus and cultured with TSt-4 cells in the absence (−) or presence (+) of 4-OHT. Genomic DNA prepared from cultured thymocytes was analyzed as described in Figure 1. The Cd3e gene was amplified as a loading control. Four-fold serial dilutions of genomic DNA from adult wild-type DN (WT DN) thymocytes and TCRβ DN thymocytes were used as controls. Data are representative of four independent experiments.
(B) PCR analysis of Vβ-Dβ signal joints in cultured thymocytes. The same sample DNAs and 2-fold serial dilutions of the control DNA used in (A) were analyzed for Vβ-Dβ signal joints by PCR. Data are representative of four independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Enforced E47 Expression Antagonizes the Pre-TCR-Mediated Feedback Signal
(A) Flow-cytometric analysis of cultured thymocytes. DN thymocytes enriched from adult wild-type (WT) and TCRβ Tg thymocytes were analyzed for surface expression of CD4 and CD8 on live-gated cells and CD44 and CD25 on lineage-negative cells (Lin: CD4, CD8, and DX5) (Preculture). WT and TCRβ Tg DN thymocytes were transduced with control virus or E47-ER-expressing virus and cultured with OP9/N-DLL1 cells in the presence of 4-OHT. Cultured thymocytes were analyzed for surface expression of CD4, CD8, and a human CD25 marker. hCD25-positive and CD4 CD8 DN or DP populations were sorted and analyzed for the verification of the purity of the sorted populations (Postculture).
(B) PCR analysis of Vβ-Dβ signal joints in cultured thymocytes. Genomic DNA prepared from presorted (Total) and postsorted hCD25-positive and CD4 CD8 DN or DP thymocytes was analyzed for Vβ-Dβ signal joints by PCR as shown in Figure 6. Two-fold serial dilutions of the control DNA used in Figure 6 were also included. The Cd3e gene was amplified as a loading control. Data are representative of three independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions